Bacillus anthracis is the cause of cutaneous, inhalation, and gastrointestinal anthrax. B. anthracis has also recently been used as an agent of bioterrorism. The identification of bacterial genes expressed uniquely in vivo during infection with B. anthracis would improve our understanding of the molecular bacterial events that occur during anthrax, and could lead to improved therapeutics and (less likely) an improved anthrax vaccine. One recently developed technique that permits identification of bacterial genes expressed uniquely in vivo is IVIAT (in vivo induced antigen technology). In this procedure, convalescent serum collected from humans or animals infected with a pathogen of interest is absorbed against bacteria grown in vitro. Absorbed serum is then used to probe a genomic DNA expression library of the pathogen of interest in an E. coli host system. Reactive clones express an antigen expressed uniquely in vivo, and reactive genes and their products can be further identified and analyzed. Our hypothesis is that B. anthracis contains genes that are expressed uniquely in vivo during anthrax, and that identification of such genes and their products will lead to improved understanding of the pathogenic events that occur during anthrax. Our specific aim, therefore, is to apply IVIAT to B. anthracis using two sets of already collected convalescent sera: one from rhesus macaques that survived inhalational challenge with virulent Ames strain B. anthracis and cleared documented B. anthracis bacteremia as part of a fully approved anthrax study at the Centers for Disease Control and Prevention; the other from humans surviving naturally acquired cutaneous anthrax (collected as part of a fully approved collaborative study between Kazakhstani and CDC researchers). We designed our study to take into account the high lethality of anthrax and the presence of a B. anthracis capsule, and in our project, we will specifically not evaluate well characterized virulence factors of B. anthracis (such as exotoxin and capsule), but will focus our efforts on identifying previously unrecognized genes uniquely expressed in vivo. IVIAT is an established protocol in our laboratory, and this preliminary collaborative study should lay a foundation for subsequent analysis of identified B. anthracis genes and their products.